scholarly journals Regulation of Hepadnavirus Reverse Transcription by Dynamic Nucleocapsid Phosphorylation

2006 ◽  
Vol 81 (4) ◽  
pp. 1641-1649 ◽  
Author(s):  
Suresh H. Basagoudanavar ◽  
David H. Perlman ◽  
Jianming Hu
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Core Protein ◽  
Early Stage ◽  
Phosphorylation Site ◽  
Duck Hepatitis ◽  
Double Stranded Dna ◽  
B Virus ◽  
Multistep Process

ABSTRACT Reverse transcription, an essential step in the life cycle of all retroelements, is a complex, multistep process whose regulation is not yet clearly understood. We have recently shown that reverse transcription in the pararetrovirus duck hepatitis B virus is associated with complete dephosphorylation of the viral core protein, which forms the nucleocapsid wherein reverse transcription takes place. Here we present a genetic study of the role of this dynamic nucleocapsid phosphorylation in regulating viral reverse transcription. Detailed analyses of the reverse transcription products synthesized within nucleocapsids composed of core phosphorylation site mutants revealed that alanine substitutions, mimicking the nonphosphorylated state, completely blocked reverse transcription at a very early stage. In contrast, aspartate substitutions, mimicking the phosphorylated state, allowed complete first-strand DNA synthesis but were severely defective in accumulating mature double-stranded DNA. The latter defect was due to a combination of mutant nucleocapsid instability during maturation and a block in mature second-strand DNA synthesis. Thus, the reversible phosphorylation of the nucleocapsids regulates the ordered progression of reverse transcription.

scholarly journals Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

2003 ◽  
Vol 278 (30) ◽  
pp. 28123-28129 ◽  
Author(s):  
Josef Köck ◽  
Michael Kann ◽  
Gerhard Pütz ◽  
Hubert E. Blum ◽  
Fritz von Weizsäcker
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Duck Hepatitis B Virus ◽  
Virus Core ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis

Hepatology ◽  
1999 ◽  
Vol 30 (1) ◽  
pp. 308-315 ◽  
Author(s):  
Fritz von Weizsäcker ◽  
Josef Köck ◽  
Stefan Wieland ◽  
Wolf-Bernhard Offensperger ◽  
Hubert E. Blum
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Core Protein ◽  
Dominant Negative ◽  
Viral Dna ◽  
Duck Hepatitis B Virus ◽  
Virus Core ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Duck Hepatitis B Virus Nucleocapsids Formed by N-Terminally Extended or C-Terminally Truncated Core Proteins Disintegrate during Viral DNA Maturation

1998 ◽  
Vol 72 (11) ◽  
pp. 9116-9120 ◽  
Author(s):  
Josef Köck ◽  
Stefan Wieland ◽  
Hubert E. Blum ◽  
Fritz von Weizsäcker
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Core Protein ◽  
Viral Dna ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
Core Proteins ◽  
B Virus ◽  
Carboxy Terminal

ABSTRACT Hepadnaviruses are DNA viruses that replicate through reverse transcription of an RNA pregenome. Viral DNA synthesis takes place inside viral nucleocapsids, formed by core protein dimers. Previous studies have identified carboxy-terminal truncations of the core protein that affect viral DNA maturation. Here, we describe the effect of small amino-terminal insertions into the duck hepatitis B virus (DHBV) core protein on viral DNA replication. All insertion mutants formed replication-competent nucleocapsids. Elongation of viral DNA, however, appeared to be incomplete. Increasing the number of additional amino acids and introducing negatively charged residues further reduced the observed size of mature viral DNA species. Mutant core proteins did not inhibit the viral polymerase. Instead, viral DNA synthesis destabilized mutant nucleocapsids, rendering mature viral DNA selectively sensitive to nuclease action. Interestingly, the phenotype of two previously described carboxy-terminal DHBV core protein deletion mutants was found to be based on the same mechanism. These data suggest that (i) the amino- as well as the carboxy-terminal portion of the DHBV core protein plays a critical role in nucleocapsid stabilization, and (ii) the hepadnavirus polymerase can perform partial second-strand DNA synthesis in the absence of intact viral nucleocapsids.

scholarly journals Evidence that the first strand-transfer reaction of duck hepatitis B virus reverse transcription requires the polymerase and that strand transfer is not needed for the switch of the polymerase to the elongation mode of DNA synthesis

2000 ◽  
Vol 81 (8) ◽  
pp. 2059-2065 ◽  
Author(s):  
Yunhao Gong ◽  
Ermei Yao ◽  
Melissa Stevens ◽  
John E. Tavis
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Reverse Transcription ◽  
Transfer Reaction ◽  
Strand Transfer ◽  
Minus Strand ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

Deletion of amino acids 79–88 in the duck hepatitis B virus reverse transcriptase had minimal effects on polymerase activities prior to the minus-strand DNA transfer reaction, yet it greatly diminished strand transfer and subsequent DNA synthesis. This mutation also reduced reverse transcription on exogenous RNA templates. The reaction on exogenous RNAs employed the phosphonoformic acid (PFA)-sensitive elongation mode of DNA synthesis rather than the PFA-resistant priming mode, despite the independence of DNA synthesis in this assay from the priming and minus-strand transfer reactions. These data provide experimental evidence that the polymerase is involved directly in the minus-strand transfer reaction and that the switch of the polymerase from the early PFA-resistant mode of DNA synthesis to the later PFA-sensitive elongation mode does not require the strand-transfer reaction.

scholarly journals Duck Hepatitis B Virus Virion Secretion Requires a Double-Stranded DNA Genome

2003 ◽  
Vol 77 (3) ◽  
pp. 2287-2294 ◽  
Author(s):  
David Perlman ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Late Stage ◽  
Viral Genome ◽  
Virus Assembly ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
Double Stranded Dna ◽  
B Virus

ABSTRACT Hepatitis B virus assembly begins with the packaging of an RNA pregenome into intracellular nucleocapsids, with subsequent reverse transcription within these nucleocapsids converting the RNA into a characteristic, partially double-stranded DNA, which, alone, is found in enveloped extracellular virions as the viral genome. Using a synchronized replication system for the duck hepatitis B virus, together with a stringent two-step assay for virion secretion, we demonstrate that this selective genome secretion results from an intrinsic secretion competence gained only by the nucleocapsids at the late stage of reverse transcription.

scholarly journals cis-Acting Sequences 5E, M, and 3E Interact To Contribute to Primer Translocation and Circularization during Reverse Transcription of Avian Hepadnavirus DNA

2002 ◽  
Vol 76 (9) ◽  
pp. 4260-4266 ◽  
Author(s):  
Karlyn Mueller-Hill ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Reverse Transcription ◽  
Template Switching ◽  
Cis Acting ◽  
Duck Hepatitis ◽  
Donor And Acceptor ◽  
B Virus ◽  
Template Switches

ABSTRACT Hepadnaviral reverse transcription requires template switches for the genesis of relaxed circular (RC) DNA, the major genomic form in virions. Two template switches, primer translocation and circularization, are required during the synthesis of the second, or plus, strand of DNA. Studies of duck hepatitis B virus (DHBV) indicate that in addition to the requirement for repeated sequences at the donor and acceptor sites, template switching requires at least three other cis-acting sequences, 5E, M, and 3E. In this study we analyzed a series of variant heron hepatitis B viruses (HHBV) in which the regions of the genome that would be expected to contain 5E, M, and 3E were replaced with DHBV sequence. We found that all single and double chimeras were partially defective in the synthesis of RC DNA. In contrast, the triple chimera was able to synthesize RC DNA at a level comparable to that of unchanged HHBV. These results indicate that the three cis-acting sequences, 5E, M, and 3E, need to be compatible to contribute to RC DNA synthesis, suggesting that these sequences interact during plus-strand synthesis. Second, we found that the defect in RC DNA synthesis for several of the single and double chimeric viruses resulted from a partial defect in primer translocation/utilization and a partial defect in circularization. These findings indicate that the processes of primer translocation and circularization share a mechanism during which 5E, M, and 3E interact.

scholarly journals Intracellular Hepadnavirus Nucleocapsids Are Selected for Secretion by Envelope Protein-Independent Membrane Binding

2000 ◽  
Vol 74 (24) ◽  
pp. 11472-11478 ◽  
Author(s):  
Hélène Mabit ◽  
Heinz Schaller
Keyword(s):  
Envelope Protein ◽  
Core Protein ◽  
Membrane Binding ◽  
Dna Viruses ◽  
Virus Particles ◽  
Duck Hepatitis ◽  
Double Stranded Dna ◽  
Selective Membrane ◽  
Membrane Attachment ◽  
Different Populations

ABSTRACT Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surface-exposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBV-transfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.

scholarly journals The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly

Virology ◽  
2006 ◽  
Vol 353 (2) ◽  
pp. 443-450 ◽  
Author(s):  
Haitao Guo ◽  
Carol E. Aldrich ◽  
Jeffry Saputelli ◽  
Chunxiao Xu ◽  
William S. Mason
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Duck Hepatitis B Virus ◽  
Virus Core ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

2002 ◽  
Vol 76 (12) ◽  
pp. 5857-5865 ◽  
Author(s):  
Xingtai Wang ◽  
Jianming Hu
Keyword(s):  
Reverse Transcription ◽  
Specific Interaction ◽  
Initial Step ◽  
Covalent Attachment ◽  
Dna Polymerization ◽  
Duck Hepatitis ◽  
B Virus ◽  
Protein Priming ◽  
Complete Protein ◽  
Two Stages

ABSTRACT Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the specific interaction between the RT and a viral RNA signal termed ε, which bears the specific template sequence for protein priming. The product of protein priming is a short oligodeoxynucleotide which represents the 5′ end of the viral minus-strand DNA and is covalently attached to the RT. We have now identified truncated RT variants from the duck hepatitis B virus that were fully active in the initial step of protein priming, i.e., the covalent attachment of the first nucleotide to the protein (RT deoxynucleotidylation), but defective in any subsequent DNA polymerization. A short sequence in the RT domain was localized that was dispensable for RT deoxynucleotidylation but essential for the subsequent DNA polymerization. These results have thus revealed two distinct stages of protein priming, i.e., the initial attachment of the first nucleotide to the RT (RT deoxynucleotidylation or initiation of protein priming) and the subsequent DNA synthesis (polymerization) to complete protein priming, with the second step entailing additional RT sequences. Two models are proposed to explain the observed differential sequence requirement for the two distinct stages of the protein priming reaction.

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